Sterols such as β-sitosterol, which are obtained as a part of non-saponified products in the refining process of vegetable oils such as soybean oil and rapeseed oil, have been known to have an action for lowering the blood serum cholesterol level. As it has recently become evident that β-sitostanol, a saturated form of β-sitosterol, has a more powerful blood serum cholesterol lowering effect than β-sitosterol, it has been receiving increasing attention.
However, since the above-mentioned free sterols and free stanols are insoluble in the micellar phase in a digestive organs, these forms are hardly appropriate for their intake to develop their physiological effects. In order to improve the fat-solubility thereof, it has been proposed to ingest them in the form of sterol fatty acid esters, and recently, it has been attempted to add sterol fatty acid esters to various food products such as margarine with phytosterols, and some of them are commercially available.
By the way, sterol fatty acid esters have hitherto not been used in foods, but used in a cholesteric liquid crystal and as a hydrophilic base material for pharmaceuticals and cosmetics.
Therefore, the sterol fatty acid esters have been produced by chemical synthesis with an acid or base catalyst. In chemical synthesis, however, the reaction is generally conducted under severe conditions; therefore several problems may arise such as degraded quality of a product and generation of undesirable by-products. In addition to that, the product might be contaminated with the by-products and the reaction catalyst. It has been unavoidable to employ highly complicated purification steps after the synthetic reaction.
In this context, the use of an enzyme such as cholesterol esterase and lipase has been recently studied.
Cholesterol esterase and lipase are both categorized as one of carboxylic acid ester hydrolases and cholesterol esterase is defined as an enzyme, which generates a free sterol and a free fatty acid from a cholesterol fatty acid ester through hydrolysis.
Lipase (which usually means triacylglycerol lipase) is defined as an enzyme, which can generate glycerol and free fatty acids from glycerol fatty acid esters through hydrolysis.
Incidentally, many enzymes are found that the enzymes have both cholesterol esterase activity and lipase activity (see D. Lombardo et al.: Biochem. Biophys. Acta., 527, (1978), 142–149, D. Lombardo et al.: Biochem. Biophys. Acta, 611 (1980), 136–146, 147–155), and even now not a few examples are known which cannot be distinctly classified whether cholesterol esterase or lipase.
The above-mentioned enzymes are known to be capable of catalyzing the hydrolysis reaction of carboxylic acid ester in common cases and, on the other hand, also capable of catalyzing the synthetic reaction of ester.
Lawrence A. et al. indicated that sterol ester hydrolase derived from canine pancreatic juice, which is known as cholesterol esterase, may catalyze the synthesis of cholesterol oleic acid esters from free cholesterols and free oleic acids (Biochem. Biophys. Acta., 231 (1971) 558–560.
D. Lombardo et al. also indicated that cholesterol esterase derived from human pancreatic juice is capable of catalyzing the synthesis of cholesterol fatty acid esters (Biochimie et al., 1980, 62, 427–432).
Myojo et al. confirmed that lipase is capable of catalyzing the synthesis of cholesterol fatty acid esters (JP 5-33712 B1).
As shown above, it has been indicated that cholesterol fatty acid esters can be synthesized using enzymes as opposed to the afore-mentioned chemical synthesis.
However, all of the conventional production examples mentioned above are referring only to the synthetic reaction for sterol fatty acid esters as a general chemical product, and are not intended for the production of sterol fatty acid esters for use as general foods, health foods or pharmaceuticals. In other words, with respect to the synthetic reaction conditions and the subsequent purification process, no consideration is given for achieving good sensory qualities including color, odor and taste, and safety of the sterol fatty acid esters, which are important factors for general foods, health foods and pharmaceuticals.
Therefore, it has been difficult to employ the sterol fatty acid esters produced in the conventional process for foods and the like.
The above-described problem exists not only in the production wherein free fatty acids is employed as the fatty acids, but also in the production wherein fats and oils containing triacylglycerol as the main component are employed as starting materials.